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R. Nakamura, A.S. Hackam; Dickkopf3 (Dkk3) is a Glial Expressed Anti–Apoptotic Wnt Pathway Protein Upregulated During Photoreceptor Degeneration . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2575.
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© ARVO (1962-2015); The Authors (2016-present)
The role of the Wnt signaling pathway in neuroprotection is poorly understood. We have demonstrated that a secreted glycosylated Wnt pathway protein, Dkk3, is upregulated in the inner nuclear layer during retinal degeneration in rd1 mice. To understand the importance of Dkk3 in photoreceptor viability we characterized Dkk3 regulation of cell survival in vitro.
HEK 293 cells were stably transfected to constitutively secrete Dkk3. Dkk3–expressing, empty vector and parental cell lines were treated with 1 µM staurosporine (STS) or 50 µM etoposide for 16 hours to induce apoptosis. Cell viability and caspase activity were measured by Wst–1 and DEVD–AMC assays, respectively. Induction of Wnt signaling by Dkk3 and the related proteins Dkk1 and Dkk2 was measured with the TOP–flash luciferase reporter assay.
HEK293 cells became resistant to STS and etoposide when Dkk3 was expressed, as shown by Wst–1 viability assays, compared with the control cell lines. Cells expressing Dkk3 had 92% viability while HEK cells with empty vector and parental cells had 37% and 40 % viability, respectively. This increased resistance to STS was associated with dramatically reduced caspase activity in Dkk3–expressing cells (unpaired t–test, p–value < 0.01). Comparable decreases in caspase activity were observed in transiently transfected SIRC (corneal epithelial) and Cos–7 (fibroblast) cells. Further, we demonstrated that Dkk3 increased Wnt signaling by 3–fold over control transfections whereas Dkk2 activated Wnt by 2–fold and Dkk1 decreased Wnt signaling 2–fold. Immunohistochemistry and RT–PCR demonstrated that rat primary retinal glial cultures express Dkk3, suggesting a hypothesis that Dkk3 may play a role in glial–regulated neuroprotection.
Overexpression of Dkk3 protects several cell lines from exogenous insults, and this protection may utilize the canonical Wnt signaling pathway. In future studies we will explore this effect in vivo and investigate the therapeutic applications of Dkk3.
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